The goal of this research is to develop HSV as a versatile and efficient gene transfer system. Gene transfer and expression of the HPRT gene will be used as a model system toward the long-term goal of gene therapy for Lesch-Nyhan disease. The project is organized into six interrelated aims: (i) Attempts will be made to engineer a completely defective nontoxic HSV vector backbone by deletion of genes which are expressed immediately on infection. The virus mutants will be propagated on complementing cell lines and characterized for the effects on neuronal cells in culture following infection. Parameters to be considered are virus multiplicity, and host cell macromolecular synthesis and viability. (ii) Nontoxic vectors (NTV) will be studied for their ability to persist long term in cell culture as extrachromosomal elements and for integration events using selectable markers. The expression of viral genes will be monitored including the latency associated transcripts using Northern and RT-PCR approaches. (iii) We attempt to direct and detect homologous segment of the integration events involving a mutant HPRT gene cloned into HSV and the wild-type HPRT gene resident in B103 cells. (iv) The HSV-NTV will be stereotactically inoculated into hippocampus and basal ganglia and examined for neuronal cell damage using histochemical approaches. Both nonimmune and HSV-immune animals will be examined to evaluate possible immunocytotoxic responses. Genome persistence and expression of viral latent and lytic genes will be examined by in situ hybridization and RT-PCR. (v) A series of experiments will be carried out to exploit the latency active promoter (LAP), strong non-HSV promoters or recombinant promoters for expression of the lacZ long-term in vitro and in vivo. Moreover, attempts will be made to design recombinant promoters responsive to the potent Gal4-VP16 transactivator which auto stimulates both its own expression and lacZ expression in the same viral genome. (vi) The HSV-NTV with a suitable promoter system developed in experiments above will be exploited to express the HPRT gene in the brains of HPRT deficient mice. Gene expression will be characterized by in situ hybridization, and enzyme production and activity in brain extracts by immuno- and biochemical assays.